Human papillomavirus (HPV) is an icosahedron DNA virus which belongs to papilloma genus A of Papillomaviridae family. The genome of the HPV is a double-stranded cyclic DNA with about 8000 base pairs, with all open reading frames (ORFs) encoded by one DNA strand. The HPV is divided into different types according to the less than 90% homology of the genomic nucleic acid sequence, the difference between the nucleic acid sequences of the virus genome in the same type at 2-10% is deemed as different subtypes, while in the same subtype, the viral genomic nucleic acid sequence difference less than 2%, is deemed as variant. Up to present, researchers have found nearly a hundred types of HPV. Different types cause different clinical manifestation and may be classified into different types according to the different tissue sites invaded: the skin-type and mucosal-type. The skin types include HPV1, 2, 3, 4, 7, 10, 12, 15 and the like, associated with common warts, flat warts, and plantar warts, etc., also include HPV 5, 8, 14, 17, 20, 36, 38, and the like associated with epidermodsplasia verruciformis. The mucosal type includes HPV6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54 and the like associate with infection in genitalia, anus, oropharynx, esophageal mucosa, and the like, and also includes HPV16, 18, 30, 31, 33, 35, 39 associated with cervical cancer, rectal cancer, oral cancer, tonsil carcinoma, etc., (Jenson A B et al. 1984, Human papillomavirus. In: Belshe R B, editor. Textbook of Human Virology. Littleton, M A: PSG-Wright; p 951-68). HPV can be classified into “high-risk” and “low-risk” types according to risk of human oncogenesis. Low-risk HPV, such as HPV 6, 11, 42, 53 and the like, are associated with infections at genital, anus, oropharynx, esophageal mucosa, and the like, and high-risk HPV is the main cause of cervical cancer and the like.
Famous German medical scientist, Harald zur Hausen, has found early in the 1970s that high-risk HPV infection is the cause of cervical cancer (zur Hausen H. 1977, Human papillomaviruses and their possible role in squamous cell carcinomas. Curr. Top. Microbiol. Immunol., 78: 1-30). His study confirms the direct correlation between the two, and thus got the Nobel prize in physiology or Medicine in 2008. Investigation on the tumor epidemiology in China by famous tumor epidemiology specialist Youlin Qiao and his team, Cancer Institute of Cancer Hospital, Chinese Academy of Medical Science discovered that the relevance between Chinese cervical disease (≥CIN2) and HPV infection can be up to 96-100% (Jing Li et al. 2013, Epidemiological Features of Human Papillomavirus (HPV) Infection among Women Living in Mainland China. Asian Pac J Cancer Prev, 14 (7): 4015-4023). Since the international workshop of the international agency for research on cancer (IARC) in 1995 HPV infection is considered the main cause of cervical cancer. The continuous infection of HPV has been considered an essential factor and main cause of cervical cancer pathogenesis. A lot of researches indicate that most cervical intraepithelial neoplasia (CIN) and almost all cervical cancer lesions have high-risk HPV infection (Remmink A J et al. 1995, The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: natural history up to 36 months. Int J Cancer, 61(3): 306-11). It is noted that, the pathogenesis of cervical cancer generally needs to undergo continuous infection of HPV to different levels of CIN(CIN1-CIN3), and eventually develops into cervical cancer (Bosch F X et al. 2002, The causal relation between human papillomavirus and cervical cancer. J Clin Pathol, 55(4): 244-65). The process is generally relatively long and lasts for 10-15 years in average, which provides sufficient time for early discovery and blocking of cervical cancer and precancerous pathology thereof.
At present, world health organization (WHO) and international agency for research on cancer (IARC) classified 14 HPV subtypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) as high-risk HPV types, and a large number of studies found that 70% of cervical cancer is caused by HPV16 and 18 (Khan M J et al. 2005, The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst, 97(14): 1072-9). Therefore, the detection of high-risk HPV 16 and 18 infection has significantly important meaning in preventing cervical cancer.
So far, HPV cannot be cultured in vitro, and as the etiology relationship in cervical cancer and high-risk HPV getting more and more attention, high-risk HPV detection has been used to evaluate the risk of cervical cancer occurrence. In order to realize early diagnosis and timely treatment to prevent the occurrence of cervical diseases (including cervical cancer), research on HPV detection is necessary.
HPV antigen detection: after the human epidermis is infected by HPV, the capsid protein is synthesized by intracellular proliferation to become HPV antigen. The immune enzyme staining can be used for detecting HPV antigen protein L1 in infected tissue cells so as to know whether HPV infection exists or not. However, since the HPV antigen immunohistochemistry method can only identify the capsid protein of the cell nucleus, while such capsid protein appears only in one stage of HPV life cycle (in late-stage virions), the antigen expression quantity differs with the degree of pathological changes. Meanwhile, the method requires a large number of virions to generate a positive reaction. Therefore, the detection rate of this method is relatively low (Dillner J. 1999, The serological response to papillomaviruses. Semin Cancer Biol, 9: 423-30).
HPV antibody detection: after HPV infects human body, the human body induces an anti-HPV antibody, so that the anti-HPV antibody in serum can be detected. The detection of antibody is mainly used for detecting the HPV16E6 and E7 antibodies. Because the detection sensitivity is low and the antibody appears at the late stage of the disease, it is not suitable for early diagnosis of the disease. In addition, after human being is infected by HPV, the antibody produced by the body can be presented for a long time, the detection of HPV antibodies by serological method cannot determine whether the infection is recent or in the past. At the same time, because there are many types of HPV and the immunoreaction is not consistent, HPV antibody detection is rarely used. (Frazer I H. 2010, Measuring serum antibody to human papillomavirus following infection or vaccination. Gynecol Oncol, 118(1 Suppl): S8-11).
Traditional morphology methods and immunological methods cannot fully prove whether HPV infection exists or not, and the clinical sensitivity and the specificity are not desirable ideal, which will induce high false positive rate and false negative rate. The newly-developed detection methods detect HPV by using a molecular biological method, including method based on signal amplification and method based on template amplification. Methods based on template amplification are based on PCR, combined with different detection technologies, mainly comprise fluorescent PCR method, PCR combining membrane hybridization method, PCR combining reverse-point hybridization method, or PCR combining quantum dot technology (Anco Molijn et al. 2005, Molecular diagnosis of human papillomavirus (HPV) infections. J Clin Virol, 32 Suppl 1: S43-51).
The PCR-based method which is based on template amplification mainly has the following problems: (1) the PCR technology is a detection method by amplifying target DNA fragments, for which the laboratory environment and the relatively strict experimental operation level are relatively high; generally speaking, more than one virus besides HPV are tested in the PCR laboratory of a hospital, so that in the process of amplifying the target DNA, a large number of different types of viral DNA amplification fragments are easily generated, these fragments can react with each other due to mistakes and operation errors of laboratory environment, so that environmental pollution of the laboratory is caused, and false positive or false negative results will occur to cause misdiagnosis or missed diagnosis of the patient; (2) many products based on the PCR technology generally only detect the L1 region of the HPV genome, and research shows that In this way, 5-10% of cervical cancer will miss diagnosis, as a result false negative results will be generated (Walboomers et al 1999, Human papillomavirus is a necessary cause of invasive cervical cancer worldwide J Pathol, 189: 12-19); (3) There is competitive inhibition in PCR reactions. When a variety of subtype infection are presented in of a patient, only the subtype with relatively high proliferation amount can be amplified, and the subtype with less virus number can result in false negative results due to relatively low amplification; that is, if the number of the virus of high-risk subtypes infected by a patient is less than the number of the virus of low-risk subtype the result will be a negative result, so that misdiagnosis of the patient is caused; (4) The sensitivity and the specificity of the detection method of the PCR technology are mainly subjected to the influence of sample transportation and preservation conditions, variation of DNA sequence in same HPV subtype, loss of HPV DNA in extraction process, primer pair design, PCR product size, and PCR program design, among others.
In conclusion, the present field needs to further optimize the HPV detection method to develop a convenient and accurate product suitable for clinical application.